Two classes of chromatography are adsorption chromatography (AC) and size exclusion chromatography (SEC). Reverse and normal phase chromatography, ion exchange chromatography, chiral chromatography are in the class of AC. Separation here is due to different energetic interactions between molecules of the substance undergoing to the chromatographic separation. Another class includes SEC that separates substances according to molecular size (other parameters affecting separation include size of pores in the media, size of the chromatographic column, flow rate, temperature, injection volume and sensitivity of used detector). Separation, using SEC, between molecules having a size difference of at least two fold is usually sufficient. Drug screening frequently requires the separation of protein-ligand complexes from non-bound ligand molecules. The complex may then need to be analyzed. If, however, short or small columns are in use, the SEC separation may be poor. If needed, additional resolution may be affected by adsorption between small molecules and the inner surface of pores of the SEC media. Generally, the interaction increases retention of the small molecules and has no effect on large molecules like proteins if they are not able to penetrate inside the pores. This variety of media is known as “restrictive access media” or RAM. Performance of the RAM is dependable on its design. As a result of using this media, complete and fast separation of protein-ligand complexes and low molecular weight compounds is achieved
Known RAM are generally not able to separate protein-ligand complexes from detergents that solubilize proteins such as membrane proteins. Frequently, the required detergent concentration results in formation of micelles comparable in size to the target protein. Existing media do not allow SEC based separation of the micelles from protein-ligand complexes. This drawback causes problems with characterizing the protein-ligand complexes, for example, using liquid chromatography-mass spectroscopy (LC-MS) screening, detergent molecules come into the mass spectrometer (MS) together with the ligands initially bound to target. The detergent molecules then suppress the ligand ionization.